Electron microscopy of viral RNA, replicative form and replicative intermediate of the bacteriophage R17

J. Mol. Biol. (1966) 22, 173-177

LETTERS TO THE EDITOR

Electron Microscopy of Viral RNA, Replicative Form and Replicative Intermediate of the Bacteriophage R17 In bacteria infeoted with RNA bacteriophages, several unique types of RNA may be found. These are the single-stranded viral RNA of molecular weight 1.1 x lo6 (Mitra, Enger & Kaeeberg, 1963; Sinha, Fujirnura & Kaesberg, 1965), double-stranded RNA termed replicatiue jomz (Ammann, Deliue & Hofschneider, 1964), and double-stranded RNA with branahed single-stranded components, termed replicaiive &termed&e (cf. Erikson & Franklin, 1966). The latter is of primary interest, since it is believed to be the double-stranded template for viral RNA with nascent single-strands. With the availability of methods of purify@ replicative form (Amman et al., 1964; Franklin, 1966a) and replicative intermediate (Franklin, 1966a), it was feasible to investigate the morphology of these various types of RNA. Fortunately, the purification of replicative intermediate coincided with the development of modifications in the Kleinschmidt technique, which allowed visualization of extended singlestrands of RNA and therefore offered a basis of comparison with replicative form and replicative intermediate (Granboulan, Huppert & Laoour, 1966). The emphasis in the present preliminary report is on the qualitative aspects of the study. More quantitative results, inoluding a more detailed analysis of the chain lengths of the various types of molecules, will be published later. Viral RNA was prepared by phenol extraction from highly purified bacteriophage R17. The virus was purified by modi6cations of procedures described by Enger, Stubbs, Mitra & Kaesberg (1963) and by Strauss & Sinsheimer (1963). These procedures are described in another paper (Vasquez, Granboulan & Franklin, 1966). Rephcative form and replicative intermediate were prepared from cells infected with bacteriophage R17 for 35 to 49 minutes. The phenol-extracted nucleic acids were separated into a 1.5 M-NaGl precipitate and a supernatant fraction. Replicative form and replicative intermediate were puri6ed by adsorption chromatography on cellulose of the salt supernatant fraction (replicative form) and the precipitate (replicative intermediate) (Franklin, 1966a). One modification has been introduced into the procedure. After the first fractionation on cellulose, the eluate containing doublestranded RNA is concentrated by the use of Ficoll (Pharmacia, Uppaala). This concentrate is re-chromatographed on cellulose and the purified double-stranded RNA is then concentrated by alcohol precipitation. The samples of RNA were prepared for electron microscopy according to the technique of Kleinschmidt, Lang, Jacherts & Zahn (1962). All samples of singlestranded viral RNA were diluted in 8 M-urea just before spreading in order to disrupt hydrogen bonds and prevent intramolecular and intermolecular tangling (Granboulan et al., 1966). The double-stranded RNA samples were examined with and without dilution in 8 ~-urea. The RNA was added to 1 ml. of cytoch.rome c or d&opropylphosphoryl trypsin (100 pg/ml.) (Stoeckenius, 1963) in ammonium acetate buffer (1.0 M, pH 8.0) containing iaopropanol (0.06%). The final concentration of 173

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RNA was between 1 and 3 pg/ml. The spreading of double-stranded RNA was improved by adding EDTA to it at a final concentration of O-02 M before mixing the RNA with the protein and ammonium acetate. Approximately 0.2 ml. of the final mixture was delivered onto the surface of ammonium acetate buffer (0.015 M, pH 8.0) in a Teflon or glass dish. The resultant film was pioked up on 300-mesh grids covered with a carbon-coated Formvar film. After drying in alcohol and isopentane, the specimens were shadowed, while rotating, with uranium oxide at an angle of 7”. The specimens were examined with a Siemens Elmiskop I at a nominal magnitlcation of 15,000 times. The actual magnification was determined and controlled with a carbon grating replica. The lengths of the molecules were measured on prints with the aid of a map measurer. Grids rich in molecules of single-stranded viral RNA from bacteriophage R17 could not be obtained. A large percentage of the molecules was probably lost in the ammonium acetate solution during the spreading process. Nevertheless, extended and unaggregated molecules could be observed in such preparations. All of these molecules were linear, i.e. non-cyclic and non-branched (Plate I(a)). Their modal length was between l-06 and 1.10 p and their mean length was 1*06&O-06~ (Fig. 1). The mole-

/

;

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I

I

I

I

II

I

0-304-l

1 0.25 %

3

-

RI7 RNA

0.20 -

z 2 0.15 g “0 0.10 c = 0.05 & e

FIG. length

Bacteriophage

---I

I 0.1

1. Single-stranded between l-06 and

11, 0.2 0.3

0.4

viral RNA: l-10 p, mean

0.5

Length length

L-rnl-lr0.6 0 7 L (length

i-l 0.8

O-9

I0

II

I2

I I3

I 4

in ~1

distribution of l-06 p f

of 90 molecules 0.06 p.

or fragmeuts.

Model

cular weight of R17 RNA is 1-l f0.1 x lo6 (Mitra et al., 1963; Sinha et al., 1966) and the ohain length is 3342 nucleotides (Siuha et al., 1966). If the mean length of 1.06 ~1 represents the complete molecule, then the base translation per residue would be 3.17 A. This value fits well with the values of 3.03 and 3.05 A determined for doublestranded RNA from reovirus (Langridge & Gomatos, 1963) and from wound-tumor virus (Tomita t Rich, 1964). The double-stranded replicative form also had a linear configuration. Unlike the single-stranded RNA, there was no change in morphology when this material was spread in the presence or absence of urea (Plate I(c)). This is undoubtedly due to the inherent lack of flexibility of the double-stranded polymer. In these preparations of double-stranded RNA, a higher percentage of fragmented molecules was found than in the single-stranded tial RNA. The lengths of the fragments oorresponded to

F 'LATE I. (a) Single-stranded RNA from bacteriophage R17, spread in presence of 8 M-U :,’ 4 5,000. replicative intermediate treated with RNase (0.08 pg/ml., 10 min, 37 (17) Double-stranded sprt ?ad in the absence of urea. x 33,750. replicative form, spread in the ahsenre of urea. Entire molecules and s’ (( :) Double-stranded frap merits can be seen. x 33,750.

P 'LATE II. (a) Replicative intermediate spread without urea. Presence the double-stranded molecules are especially prominent where indicated “&l‘l iation in the branch point and in the length of the branch. ( + 1) Long coiled on itself. x 45,000. end of the molecule intermediate spread in presence of 8 xx-urea. Arrow (11) Replicative p-0’ minent branch attached to the double-stranded “hackbone” molecule.

of branches attached to by arrows. Not,e 1the branch attached t,o cme indicates an x 45,000.

especia

;lly

LETTERS

TO

THE

EDITOR

176

quarter and half the length of the entire molecule (mean length of 1.07 p and modal length between 1.05 and 1.10 p). These maximum lengths fit well with the molecular weight of replicative form of a.pproximately 2-Ox lo8 (E&son, Fenwiok & Frsnklin, 1965). Reasoning that the inteot double-strand is comprised of two complementary molecules of chain length 3342 nuoleotides, the expected length would be 1.01 to 1.02 p if we again assume the translation per residue of 3.03 to 3.05 A (Langridge & Gometos, 1963; Tomita & Rich, 1964). The linear configuration and the length of these molecules corresponded to those found for the replicative form of the RNA phage M 12 (Ammann, Delius, & Hofschneider, 1964). Replicrttive intermediate, spread either in the presence or absence of urea, had the morphology of a branched linear polymer (Plate II (a), (b)). Most frequently there was only one branch, rarely two, very rarely three. The position of the branch point along the molecule, as well as the length of the branch, were varis,bles (Plate II (a), (b)). Sometimes the branch was at one end of the molecule. When this ocourred, the branch was usually coiled on itself end it was not feasible to determine the length of the coiled part (Plate II (a)). The length of the “backbone” molecule was similar to that of replicative form and the presence of fragmented molecules was slso noted. The lengths of the fragments corresponded to quarter and half of the maximum length, as can be seen from the histogram of length distribution (Fig. 2). In a popu-

II

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36

0.25

% 2

0.20-

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-

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I

I

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I

I I.3

I I.4

intermediate

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intermediate Three peaks: molecules).

spread in presence 1 to 1.06 p (entire

0 8

0.9

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of 8 ~-urea: length distribution of 266 molecules); 0.50 to O-65 p (half molecules);

lation of replicative intermediate, intact molecules with visible branches comprised about 40% of a population of 238 intact molecules. Molecules are considered intact if their lengths fall within the modal length. This value was fairly constant for different samples of replicetive intermediate, with spreading in the presence or absence of urea. There is between 20 and 30% single-stranded component in a population of replicative intermediate (Franklin, 1966a). The single-stranded component may be considered a population of linear polymer molecules in a steady-state of polymerization with the special condition of finite chain termination (Franklin, 1966, manuscript in preparation). From the distribution function for single-strands, we can calculate the number of single-strands per double-stranded molecule to lie

176

N. GRANBOTJLAN

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R. M. FRANKLIN

between O-50 (for 20% single-stranded component) and 0.86 (for 30% single-stranded component). Assuming a Poisson distribution of single-strands per double-stranded template, the expected percentage of intact double-stranded molecules with no single-strands should lie between 6O-6o/oand 42.3%) which fits well with the observed value just mentioned. When such samples of replicative intermediate were digested with RNase (O-08 pg/ml., 10 min, 37”C, followed by phenol extraction to remove the RNase), the spread molecules had the same morphology as those of replicative form and the majority of branches had disappeared (Plate I(b)). Under these conditions, only 6% of the intact molecules were branched. In a control experiment, equal amounts of single-stranded viral RNA and double-stranded replicative form were mixed and studied by the Kleinschmidt technique. No branched molecules were seen in such preparations. The branched molecules found only in preparations of replicative intermediate, the removal of the branches with RNase, their absence in the replicative form, and the invariance of the replioative intermediate “backbone” in the presence or absence of urea, confirm the biochemical and biophysical data, i.e. that replicative intermediate is characterized by single-stranded “tails” or branches hydrogen-bonded to the double-stranded RNA molecule (Erikson & Franklin, 1966; Franklin, 1966a,b). The branch often appears extended even when replicative intermediate is spread in the absence of urea. This could possibly result from electrostatic repulsion between the branch and the backbone. On the other hand, one can find examples of extreme coiling of the branch in the absence of urea, especially when it is located at the end of a chain. This is further evidence for the single-stranded character of the branch component. We sre much indebted to Miss Elizabeth Hinckley and Miss Marianne Salditt for their excellent assistance in the preparation of virus and the puritlcation of the nuoleio acids, to Mr Alain Niveleau for assistance with the electron microscopy, and to Drs L. V. and E. H. Crawford for the gift of diisopropylphosphoryl trypsin. Laboratoire de Microscopic Electronique Institut de Recherches sup le Cancer Villejuif 94 (Val de Marne) France and Department of Pathology University of Colorado School of Medicine Denver, Colorado 80220, U.S.A. Received

18 July

t Present address: of New York, Inc.,

NICOLE GRANBOULAN RICHARD M. FRANKLINt

1966 Department New York,

of Virology, New York

The

Public

Health

Research

Institute

of the

City

10009, U.S.A.

REFERENCES Ammann, J., Delius, H. & Hofscbneider, P. H. (1964). J. Mol. BioZ. 10, 667. Enger, M. D., Stubbs, A., Mitra, S. & Kaeeberg, P. (1963). Proc. Nat. Acad. Sci., Wa&. 40, 867. Erikson, R. L., Fenwick, M. L. & Franklin, R. M. (1966). J. Mol. BioZ. 13, 399. Erikson, R. L. & Franklin, R. M. (1966). Bad. Rev. 80, 267. Franklin, R. M. (1966a). PTOC. Nat. Acad. Sci., Wash. 55, 1504. Franklin, R. M. (1966a). A&t. Second Int. Biophysics Congrem, Vienna, Vienna: Gistel & Cie. Granboulan, N., Huppert, J. BELacour, F. (1966). J. Mol. BtiZ. 16, 671.

LETTERS

TO

THE

EDITOR

Kleinschmidt, A. K., Lang, D., Jacherts, D. & Zahn, R. K. (1962). Biochina. bio-phye. 61, 857. Langridge, R. & Gomatos, P. J. (1963). Science, 141, 694. Mitra, S., Enger, M. D. & Kaesberg, P. (1963). Proc. Nat. Acud. Sci., Wcmh. 50, 68. Sinha, N. K., Fujimura, R. K. & Kaesberg, P. (1965). J. Mol. Biol. 11, 84. Stoeckenius, W. (1963). Appendix to We& R. & Vinograd, J., Proc. Nat. Acad. Sci., 50, 130. Strauss, J. H., Jr. Bt Sinsheimer, R. L. (1963). J. Mo2. Biol. 7. 43. Tomita, K. & Rich, A. (1964). Nature, 201, 1160. Vasquez, C., Granboulan, N., & Franklin, R. N. (1966). J. Bad. (in the press).

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